Cannabis is today the most widely abused illicit drug in the world (Journal of Chromatography B, volume 733, pages 119-126) and in many countries it is assigned as a class 1 or class 2 prohibited drug. The detection of cannabinoids is routinely carried out by forensic scientists, toxicologists, drug rehabilitation clinics and as part of workplace testing for drugs of abuse. Recently, it has become a major illicit drug targeted for driving under the influence testing.
Delta-9-tetrahydrocannabinol (THC, see structural diagram I in FIG. 1) is the primary psychoactive analyte in the flowering or fruity tops, leaves and resin of the plant Cannabis sativa. THC is extensively metabolised in man to two main products, 11-nor-9-carboxy-delta-9-THC (THC-COOH, see structural diagram II in FIG. 1) and 11-hydroxy-delta-9-THC (THC-OH, see structural diagram III in FIG. 1). Of these two metabolites, only the hydroxy metabolite has psychoactive effects.
Known THC detection methods, and known cannabis detection methods in general, can be classified into three groups. The gold standard techniques employ GS/MS, tandem GC/MS/MS, LC/MS and tandem LC/MS/MS (GC=gas chromatography; LC=liquid chromatography; MS=mass spectrophotometry). These techniques are strictly laboratory based and require specialist training and equipment, which renders them unsuitable for point-of-care testing.
Qualitative methods for cannabis detection are also known in the prior art. U.S. Pat. Nos. 3,715,189, 4,196,167, 4,771,005, 4,816,415 and 5,457,054, and also GB Patent no. 1426177 and references within, describe methods for cannabis detection based on chemical reactions involving colour change. However, chemical methods tend to be non-specific and lack the sensitivity level required for the detection of drugs in biological matrices.
The third group of known cannabinoid detection methods employ immunological techniques. For example, antibodies are raised to various cannabinoid derivatives conjugated to a carrier macromolecule and immunoassays are then set up using these antibodies and labelled derivatives thereof, e.g., radiolabelled or fluorescent derivatives for use in radioimmunoassays and fluoroimmunoassays and enzyme-labelled derivatives for use in various formats of enzyme immunoassay. Such enzyme immunoassays include, for example, direct ELISAs using immobilised drug derivative and enzyme-labelled antibody. As an alternative format, labelled anti-cannabinoid antibodies, e.g. such antibodies labelled with latex particles or colloidal gold, have been used for cannabinoid detection by lateral flow immunochromatography. The drug derivatives used are generally based upon the parent molecule delta-9-tetrahydrocannabinol (THC) and its major metabolite THC-COOH. For THC modification, a cross-linking reagent is normally attached to the compound at the C2, C5′ or the phenolic group C1 position (see for example patents GB1364925, EP0276732, EP0279308, U.S. Pat. No. 5,237,057, U.S. Pat. No. 5,747,352 and Nature New Biology (1972) volume 236 pages 216-217). THC-COOH can also be derivatised in the same manner as THC. In addition, the availability of the carboxylic group at C9 of THC-COOH allows direct cross-linking to the amino groups of carrier molecules using the carbodiimide coupling reaction. Antibodies raised to THC or THC-COOH coupled via the C1, C2 and C5′ are expected to show different cross-reactivity for those two compounds due to the absence and presence of the carboxylic group at C9, respectively. Therefore, to achieve comparable cross-reactivity for THC and THC-COOH, many of the known immunoassays for detection of those compounds employ antibodies raised to THC-COOH coupled to a carrier molecule via the carboxylic group at C9.
Many immunoassays for THC are commercially available. The Roche Diagnostics radioimmunoassay (U.S. Pat. No. 4,438,207) and fluorescence polarisation immunoassay (European Patent no. 0392332) use antibodies raised to THC-COOH coupled to a carrier protein via the phenolic group at C1 and a similarly radiolabelled or fluorescence labelled THC-COOH. As expected, this assay shows excellent detection of the acid but only 5% cross-reactivity with the parent drug, THC. Abbott's fluorescence polarization immunoassay (European Patent no. 0279308) uses antibodies raised to THC-COOH linked to a carrier protein via the carboxylate group at C9 and THC-COOH with a fluorescent moiety at the same position. This assay shows a better cross-reactivity with the parent drug as well as the 11-hydroxy derivative (FIG. 1, compound III). A number of enzyme immunoassays for cannabinoid detection are also available including one made available by Cozart Bioscience Limited. These employ an antibody raised to THC-COOH linked to a carrier protein via the carboxylate group and horseradish peroxidase (HRP)-labelled THC-COOH (label attached via the carboxylate group of the acid).
Almost all of the described immunoassays are good for detecting THC-COOH making them suitable for the detection of cannabis use using matrices like urine and blood. However, oral fluid (saliva) from cannabis users shows no or minute amounts of THC-COOH and THC-OH. Determination of the primary psychoactive component, THC, is thus essential in the case of roadside testing for driving under the influence of that drug using oral fluid samples (proposed as the best matrix for roadside testing for drugs of abuse). The ability to detect low amounts of the parent drug THC is also necessary to be able to pick up recent use of this drug. The present invention addresses this problem by providing new competitive immunoassay techniques for THC detection, which may also provide the advantage of exhibiting high cross-reactivity for both THC-COOH and THC-OH.